The complement system is a complex group of proteins in body fluids that, working together with antibodies or other factors, play an important role as mediators of immune, allergic, immunochemical and immunopathological reactions. Activation of the complement system can result in a wide range of reactions such as lysis of various kinds of cells, bacteria and protozoa, inactivation of viruses, and the direct mediation of inflammatory processes. Through the hormone-like activity of several of its components, the complement system can recruit and enlist the participation of other humoral and cellular effector systems. These in turn can induce directed migration of leukocytes, trigger histamine release from mast cells, and stimulate the release of lysosomal constituents from phagocytes.
The complement system consists of at least twenty distinct plasma proteins capable of interacting with each other, with antibodies, and with cell membranes. Many of these proteins when activated combine with some of the other proteins to form enzymes to cleave and activate still other proteins in the system. The sequential activation of these proteins follows two main pathways; the classical pathway and the alternative pathway. Both pathways use a common terminal trunk which leads to cell lysis or virus inactivation.
The classical pathway can be activated by antigen-antibody complexes, aggregated immunoglobulins and non-immunological substances such as DNA and trypsin-like enzymes. The classical pathway of activation involves, successively, four components denominated C1, C4, C2 and C3. These components can be grouped into two functional units: C1 or recognition unit; and C4, C2, and C3 or activation unit. Five additional components denominated C5, C6, C7, C8, and C9 define the membrane attack unit forming the terminal truck common to both pathways
In the classical pathway, C1 is activated such as by attachment to an immunoglobulin, and through a series of reactions, produces an activated Cls from a constituent of Cl. A bar over the term for a complement factor denotes an active enzyme. Activated Cls cleaves portions of both of components C4 and C2. Parts of the C4 and C2 components then combine to form the activated complex C4b,2a having a molecular weight of about 280,000. C4b,2a is a proteolytic enzyme which continues ongoing complement action. Earlier components are no longer required after it has been formed. C4b,2a cleaves and thereby activates the next component of the sequence, C3, to produce C3b which attaches to cell membranes adjacent to the C4b,2a. The C3b then combines with the C4b,2a to form the last activated complex in the classical pathway C4b,2a,3b. This enzyme cleaves C5, a component of the membrane attack unit.
The alternative pathway, also known as the properdin pathway, comprises at least six components. Five of these components truly belong to the alternative pathway, factors B, D, properdin (P), and two inhibitors, H and I. The sixth component, C3, can also be found in the classical pathway. Component C3b is sometimes also known as factor A. The alternative pathway can be activated by immunological substances such as IgA and non-immunological substances such as certain complex polysacharides, trypsin-like enzymes and cobra venom factor. Even in the absence of any antibody or immunoglobulin, the alternative pathway can destroy microorganisms.
Activation of the alternative pathway proceeds in a different manner than activation of the classical pathway. An initial requirement is the presence of C3b which appears to be continuously generated in small amounts in the body. C3b production is thought to be due to water induced cleavage of a thioester bond in C3 forming an activated C3* which reacts with the factors B and D to generate an enzyme to cleave C3 into C3a and C3b. C3b can be further produced by a positive feedback mechanism in which factor D and Bb (a component of factor B) combine with C3b to form the activated complex C3b,Bb that acts as an enzyme in an amplification loop to cleave more C3 to form additional C3b. Factors I and H act as regulator proteins by cleaving C3b to render it inactive. Other regulator proteins include C1 inhibitor and C4 binding protein.
C3b,Bb enzyme molecules are rendered more efficient by properdin (P) which binds to the complex and stabilizes it by slowing the spontaneous dissociation of factor Bb. Both C3b,Bb and C3b,P,Bb cleave additional C3 molecules to form modified poly-C3b enzymes, C3b.sub. n,Bb/ and C3b.sub. n,P,Bb,/ wherein "n" is greater than 1. Any of these molecules can also cleave C5 into C5a and C5b and initate the membrane attack unit of the same common terminal trunk. The C5b then combines with C6 and C7 to form an active trimolecular complex, C5b,6,7. The C5b,6,7 then combines with C8 and a plurality of C9's to form a further, active complex, which on the surface of a cell causes cytolysis.
When either pathway of the complement system is activated, the component C3 is proteolytically cleaved into C3a and C3b. C3b, through an ester bond, can link to biological membranes or particles. C3b also cooperates with other components in the complement system such as factor B and properdin or C4b and C2a to activate the membrane attack complex of components C5 though C9.
C3b can be proteolytically cleaved by factors H and I together, or by factor I alone where the C3b is bound to a type 1 complement receptor (CRl), to generate the inactivated molecule iC3b. The iC3b molecule can then go through several degradations to form C3d, C3c and C3d,g, also known as alpha-2D. C3d,g can be cleaved to form C3d and C3g with the ester bond discussed above being on the C3d molecule. In the case of cell-bound iC3b, the degradation products C3d,g and C3d remain bound to the cell by this ester bond.
Immune complexes (ICs), composed of antigens and their respective antibodies, appear to be involved in the pathogenesis of a diverse array of human and animal diseases. These include autoimmune, infectious (bacterial, parasitic, viral), neoplastic and other unclassified disorders. The primary means by which immune complexes mediate tissue injury is activation of the complement system, resulting in release of biologically active peptides (C3a, C3dg, C5a). Along with immune complex-fixed C3 fragments, these peptides induce such biologic phenomena as immune adherence, leukocytosis, chemotaxis and release of injurious mediators and of proteolytic enzymes.
Immune complexes and C3-C5 fragments also appear to exert profound effects on a variety of immune functions, both humoral and cellular. These effects may enhance or suppress immunity depending primarily on the antigen to antibody ratio, the isotype of antibody involved, and the balance between C3 and C5 fragments. Therefore, there is a great interest in developing techniques for detection and quantitation of immune complexes and products of activated complement components. A long list of assays for immune complexes now exists, but none is specific or sensitive enough to have the diagnostic and/or prognostic value required.
U.S. Pat. No. 4,342,566 to Theofilopoulos et al. describes an assay in which solid phase-bound polyclonal anti-human C3 reacts with a test sample, and bound complement-fixing immune complexes are subsequently detected with a radiolabeled or enzyme-linked antibody to human IgG or with radiolabeled protein-A from Staphylococcus. The anti-C3 is used in its F(ab').sub.2 form to avoid false positive results caused by rheumatoid factors (anti-homologous and/or heterologous-gamma-globulin autoantibodies) that might be present. Owing to the lack of exclusive specificity for C3c or C3d of the original polyclonal anti-C3 antibody employed, the assay has not subcategorized the complement-fixing immune complexes detected according to the state of C3 that they carry (C3b, iC3b, C3d). Competition between immune complex-bound C3 fragments and nonactivated free C3 might have also reduced the assay's sensitivity.
Study and measurement of the activation of a complement pathway can provide an indication of many possible biological disorders. The two complement pathways have been implicated in the pathogenesis or symptomatology of a broad spectrum of human diseases and pathologic conditions. In the case of the classical pathway, these include immune complex diseases of several types, autoimmune diseases, in particular systemic lupus erythematosus, and infectious diseases. The alternative pathway has been found to be involved in infections with gram negative bacteria, viruses, parasites, fungi, gram negative septicemia, and various dermatologic, renal, and hematologic diseases. Alternative pathway activation has also been associated with trauma, burns and adult respiratory distress syndrome (ARDS), as well as contact with dialysis membranes such as during hemodialysis and cardiac bypass surgery. In vitro studies have indicated that a number of gram negative bacteria and bacterial products, virus infected cells, viruses, protozoa, fungi, burns, damaged and injured cells, and other substances of biomedical importance have the ability to activate the alternative pathway in human serum.
Further background information on the operation and measurement of the complement system can be found in Cooper, "The Complement System" in Basic and Clinical Immunology, pp. 124-135, Stites et al. editors, Lange Medical Publications, Los Altos, CA. (1982); H. Rapp and T. Borsost, Molecular Basis of Complement Action, pp. 81-83, Appleton Century Crafts, New York, N.Y. (1970); Muller-Eberhard, et al., Adv. Immunol., 29:1-53 (1980); Pangburn et al., J. Immunol., 124:977-982 (1980); Schreiber et al., Clin. Immunol. and Immunopathol., 15:384-396 (1980); Platts-Mills et al., J. Immunol., 113:348-357 (1974); Lesavre et al., J. Immunol., 123:529-534 (1979); Polhill et al., J. Immunol., 121:363-370 (1978); Fearon et al., J. Immunol., 115:1357-1361 (1975); Day et al., Scand. J. Immunol., 5:715-720 (1976); Chapitis et al., J. Exp. Med., 143:241-257 (1976).